Russenkaninchen rabbits strain (Crl:CHBB (HM)) were from Charles River Wiga GmbH (Sulzfeld, Germany). Male (2.4 – 3.2 kg, ca. 50 weeks) and female rabbits (2.6 – 3.0 kg,) were housedunder 12 h light : 12 h dark (lights on 7:00 h) at 22°C and had access to standardchow (Ssniff, Soest, Germany) and water ad libitum., Only one strain of rabbits was used because of the high costs of miglustat. Also for this reason the Russenkaninchen strain was used because of the relatively small size of the animals of this strain.

Results

Miglustat has no effect on fertility in male rabbits

Miglustat uptake did not affect the bodyweight and food intake of the rabbits (data not shown). Each of the 5 male rabbits in the control group and in the 50 mg/kg/day group were mated to one female each after 8 weeks of miglustat treatment. All of the 5 drug-treated rabbits were fertile, comparable to the rabbits of the control group (Table1). Also, the litter size was not affected by miglustat treatment (Table 1).

Table 1

Species

Strain

Pregnancies/male

Pups/litter

Control

Miglustat

Control

Miglustat

Rabbit

Russen-Kaninchen

1.0 ± 0.0

1.0 ± 0.0

7.0 ± 1.2

7.2 ± 1.5

Mouse

C57BL/6

2.6 ± 0.9

0.0 ± 0.0*

6.7 ± 1.0

N/A

129S1/SvImJ

1.6 ± 0.5

1.6 ± 0.5

8.2 ± 0.6

8.9 ± 1.9

FVB/N

2.6 ± 1.5

2.2 ± 1.5

6.7 ± 1.6

6.0 ± 0.7

DBA/2

3.0 ± 0.7

2.8 ± 1.1

7.5 ± 0.8

6.3 ± 1.3

AKR/J

1.8 ± 0.4

1.4 ± 0.5

6.2 ± 2.3

3.6 ± 1.5

MA/MyJ

1.6 ± 0.5

0.8 ± 0.8

7.8 ± 0.8

3.8 ± 3.3

Effects of miglustat administration on the fertility of male mice and rabbits as assessed by natural mating.

Sperm motility (%) and morphology in rabbits. A. Spermatozoa were released in medium and percentage motile spermatozoa was estimated by counting flagellating or non flagellating spermatozoa. B. For assessment of morphology, a smear was air-dried on a slide and stained with PNA and DAPI and counted manually for abnormal sperm heads. Data are presented as the mean ± SD of percentage motile spermatozoa for 5 males and as the mean ± SD of percentage abnormal spermatozoa. Nuclear morphology and acrosomal staining were assessed by examining 100 spermatozoa per rabbit.

Sperm morphology and motility in multiple mouse strains

In ourprevious studies [10-12], we used animals from the C57BL/6 strain. Here, we administered the drug to males of 18 additional strains other than C57BL/6. According to the genealogy of inbred strains of mice [18], 11 of the strains used were from the Swiss (FVB/N) and Castle (129S1/SvImJ, A/J, AKR/J, BALB/c, C3H/HeN, CBA/Ca, DBA/2, NZB, NZW and SM/J) families of strains, 6 from the C57 lineage (C57BL/10, C57BR, C57L, C58, MA/My and YBR), and one strain had a mixed background (MRL/Mp). After treating these mice with 150 mg/kg/day miglustat (a dose that renders C57BL/6 mice infertile), we examined the acrosomal status and nuclear shape of their cauda epididymal spermatozoa, by indirect immunofluorescence with an anti-acrosomal monoclonal antibody and a nuclear dye. The effects of the drug on sperm phenotypevaried between mouse strains (Fig. 3). Because the vastmajority of C57BL/6 spermatozoa are without an acrosome, and have a strongly abnormal (non-falciform) nuclear morphology after miglustat administration [10], we firstscored the spermatozoa of the other strains for these features. This analysis divided the mouse strains into three groups. Firstly, five strains showed a major (>75%) drug-induced reduction in the percentage of acrosome-bearing spermatozoa and a high (>75%) increase in the proportion of sperm cells with non-falciform sperm nuclei (C57BL/6, C57BL/10, C57BR, C57L/J and C58/J, Fig. 4A). Secondly, three strains had moderate (15–30%) changes in both of these sperm parameters (BALB/c, AKR/J and MA/MyJ, Fig. 4A). Finally, in most strains the drug did not increase the proportion of spermatozoa with grossly misshapen nuclei, while the two most-affected strains of this third group (MRL/Mp and YBR) had a 20–25% reduction in the appearance of acrosomes (Fig. 4A). Thus the mice with the most perturbed sperm phenotype were from 5 closely related strains of the C57 family. The other two strains of this lineage were either moderately (MA/MyJ) or slightly (YBR) affected, according to the criteriaapplied.

Quantitation of effects of miglustat on morphology of spermatozoa from inbred mouse strains and interstrain hybrid mice. Effects of miglustat administration on morphological features of cauda epididymal spermatozoa from (A and B) mice of various inbred strains (n = 2–3 per strain) and from (C and D) fourth-generation C57BL/6 × FVB/N hybrid mice. In (A and C) spermatozoa were scored for possession of an acrosome (present/absent), irrespective of acrosomal staining pattern, and for possession of a grossly abnormal non-falciform nucleus. In (B and D) spermatozoa were scored for acrosomal staining pattern (normal/abnormal), and for the normality of their nuclear morphology. Falciform nuclei (flat and curved) that deviated from the typical shape of spermatozoa from untreated mice were score as abnormal. In (C and D) data are presented from the hybrid mice that were most affected by miglustat, and from a representative number of hybrid mice that showed a lesser response. Each datapoint in (A and B) expresses the difference between the average score of drug-treated mice from one inbred strain and the average score of control mice of the same strain. Each datapoint in (C and D) expresses the difference between the score of one drug-treated hybrid mouse and the average score of untreated FVB/N mice. Data points were fitted by linear regression; the corresponding trend lines are displayed in grey (correlation coefficients in A, B, C and D were 0.97, 0.79, 0.98 and 0.84, respectively). Miglustat administration was at 150 mg/kg/day, except SM/J mice (15 mg/kg/day). At least 200 spermatozoa per mouse were scored for nuclear morphology and acrosomal staining.

The sperm phenotype of miglustat-treated FVB/N mice was furthercompared with that of the C57BL/6 strain. To complement the data on acrosome status obtained by immunostaining with monoclonal Mab18.6, we used western blotting to compare the levels of two other acrosomal components, a protein present in the acrosomal matrix (sp56, [17]) and a protein localized on the inner acrosomal membrane (IAM38, [16]). After administration of miglustat to C57BL6 mice neither sp56 nor IAM38 was detectable in their spermatozoa, while in drug-treated FVB/N mice the levels of these acrosomal proteins were comparable to those of untreated animals of this strain (Fig. 5). Thus the percentage of spermatozoa that stained with Mab18.6 after miglustat treatment (2.9% for C57BL/6 and 92% for FVB/N mice) correlated very well with the levels of sp56 and IAM38 as measured by western blotting.

Figure 5

Effects of miglustat on acrosomal proteins as detected by western blotting. The impact of miglustat administration on the levels of the acrosomal proteins sp56 and IAM38 was assessed both in C57BL/6 and in FVB/N mice. Replicate western blots were prepared with lysates of epididymal spermatozoa and probed with antibodies against sp56, IAM38, or cytochrome C oxidase subunit I. Miglustat treatment was at 15 mg/kg/day.

Furthermore, after treatment of FVB/N mice with a higher dose of miglustat (600 mg/kg/day) the appearance of their spermatozoa was comparable to that seen at 150 mg/kg/day (Fig. 6B and Fig. 3E, respectively). Both at 150 and 600 mg/kg/day the drug-induced abnormalities were limited to mild nuclear and acrosomal irregularities. The percentages of spermatozoa with these defects did not differ significantly between the lower and higher drug dose (data not shown).

Miglustat has a variable impact on the fertility of male mice

To establishwhether the miglustat-induced alterations in the sperm phenotypes from the Swiss/Castle mice were relevant for their fertility, drug-treated males (150 mg/kg/day) from a number of these strains were assessed in a natural mating test. The strains used in this test were selected to represent the extent of variation in the responses to miglustat among the Swiss/Castle strains (Fig. 4B), from slight (129S1/SvlmJ), moderate (FVB/N), to more profoundly affected (AKR/J and DBA/2J). In addition, the moderately impacted C57 strain MA/MyJ (Fig. 4A) was also tested. In contrast to miglustat-treated C57BL/6 mice, the fertility of the 129S1/SvlmJ, FVB/N and DBA/2J males was unchanged (Table 1). Miglustat treatment reduced the size of litterssired by the AKR/J and MA/MyJ males, but this was not statistically significant (Table 1). Thus the considerable increase (69%) in the percentage of spermatozoa with a misshapen acrosome, in combination with moderately more (37%) spermatozoa with an abnormal falciform nuclear morphology, did not impair the fertility or size of litters sired by male mice, as seen in the DBA/2J strain. Similarly, with a 29% increase in spermatozoa without an acrosome and 16% of sperm cells with a severely abnormal (non-falciform) nuclear shape, the AKR/J were not infertile. Therefore, whereas miglustat affected the sperm phenotype in many mouse strains (Fig. 3 and Fig. 4A and 4B), this did not result in infertility in most cases (Table 1). Clearly, drug-induced infertility (as seen in C57BL/6 mice) was associated with a major absence of acrosomes and high proportion of grossly aberrant sperm nuclei.

Miglustat levels in serum in mouse strains and rabbits

The variation in the effects of miglustat among rabbits and different mouse strains could be due to unequal serum levels, which, in turn, may be a reflection of variability in the rate of renal drug excretion. Therefore, miglustat levels in serum were determined by LC/MS. Although administration of 15 mg/kg/day miglustat s.c. via mini osmotic pumps rendered C57BL/6 but not FVB/N mice infertile, the drug treatment generated a similar miglustat level in the serum of C57BL/6 and in FVB/N mice (Table 2). In rabbits the serum level was determined at the highest dose used (50 mg/kg/day), at which the animals were fertile. The serum level found in rabbits exceeded the level in C57BL/6 mice 17-fold (Table 2). Thus, the serum concentration of miglustat did not correlate with the fertility status of the mice and rabbits.

Table 2

Species

Mouse

Rabbit

Strain

C57BL/6

FVB/N

Russen-kaninchen

Dose (mg/kg/day)

15

15

50

Serum level (μM)

0.49 ± 0.26

0.61 ± 0.17

8.39 ± 5.38

Fertility (fertile/total)

0/6

6/6

5/5

Serum levels of miglustat in selected strains/species.

Genetic basis of the susceptibility to miglustat

Having observed that the consequences of miglustat administration varied between mouse strains, we reasoned that the differential response to the drug possibly has a genetic basis. Therefore, we generated small numbers of second-generation interstrain hybrid mice from the C57BL/6 and FVB/N strains. Eight F2 hybrids were obtained from C57BL/6 males and FVB/N females, and 6 F2 hybrids were bred from FVB/N males and C57BL/6 females. In each of these two groups of F2 hybrid mice one animal displayed the C57 drug-induced sperm phenotype and one animal had a phenotype that was intermediate between the two original strains. The remainder of the F2 hybrids responded to miglustat administration in the same way as FVB/N mice (data not shown). Thus, the high sensitivity to the imino sugar (resulting in the production of acrosome-less non-falciform abnormal spermatozoa) had both a maternal and a paternal inheritance.

To further investigate the genetic basis of the imino sugar responsiveness, we generated over 200 fourth-generation hybrid mice from C57BL/6 males and FVB/N females. These animals were treated with 15 mg/kg/day of miglustat, and their epididymal spermatozoa were examined and scored for the acrosomal and nuclear parameters described above. Over 70% of the hybrid mice responded to miglustat administration in a similar fashion as the FVB/N mice, while only 3 of them behavedlike a C57BL/6 mouse. However, the remainder of the interstrain hybrid mice showed a range of effects, from low to very high percentages of non-falciform dysmorphic spermatozoa without acrosomes (Fig. 4C and Fig. 6C–F). Among the hybrid mice were a number of animals with an intermediate drug-induced phenotype (between 30 and 70% reduction in acrosome presence, and between 20 and 70% increase in non-falciform abnormal nuclei, Fig. 4C). Such a phenotype was not seen in any of the inbred mouse strains after miglustat treatment (Fig. 4C vs. 4A). Similar to the inbred strains, the epididymal spermatozoa from the interstrain hybrids also displayed irregular acrosomal structures and atypical falciform nuclei (Fig. 4D and Fig. 6C–F).

redgrey

Discussion

We have evaluated the reproductive effects of miglustat in rabbits and in various strains of the C57 and Swiss/Castle lineages of inbred mice. In contrast to miglustat-treated C57 mice, fertility was not affected in the other mouse strains and the rabbit strain that have been studied. The contraceptive effect of miglustat seems therefore to be specific to the C57 strains.

In mice, the most profound effects of miglustat on the shape of sperm nuclei and on the presence of acrosomes on sperm heads were found in strains of the C57 family (the majority of sperm nuclei non-falciform dysmorphic, most spermatozoa without an acrosome), correlating closely with the contraceptive action in C57BL/6 mice. A milder category of sperm aberrations after miglustat treatment was found in most strains of the Swiss/Castle lineages (low to moderate frequencies of mild morphological abnormalities of falciform nuclei, most spermatozoa with acrosomes, a fraction of them with imperfections or aberrations). Clearly, this type of spermatozoal abnormalities is not of a severity that impairs the fertility of the animals, as the drug-treated 129S1/SvImJ, FVB/N and DBA/2 males had normal pregnancyrates in mating tests. We also observed an intermediate level of sperm aberrations, in miglustat-treated BALB/c, Ma/MyJ and AKR/J mice (a minority of spermatozoa with non-falciform nuclei, without acrosomes). This level of miglustat-induced spermatozoal abnormalities was not associated with infertility in the drug-treated Ma/MyJ and AKR/J mice. Finally, in one rabbit strain miglustat administration did not result in any observable changes in sperm phenotype.

What therefore underlies the differences in the effect of miglustat between various mouse strains and between species? One possibility is that the males of the Swiss/Castle strains and the rabbits could be less sensitive to the drug. That wouldimply that a higher dose of miglustat should result in a more severe phenotype in these strains and in rabbits. We have investigated this in FVB/N mice, by administering 150 and 600 mg/kg/day of miglustat. We found the same effects in males of this strain at both doses of the drug, not a C57-style sperm phenotype at the higher dose. This suggests that it is unlikely that the disparity in the imino sugar-response between Swiss/Castle and C57 strains is due to a lower drug sensitivity of the former strains, rather that the consequences of miglustat in these strains are qualitatively different. Secondly, the differences in reproductive outcome of miglustat treatment observed between mouse strains and species could be due to differential pharmacokinetics of the imino sugar (e.g. variations in the rate of renal excretion), resulting in different serum concentrations when administered at the same dose of drug. Clearly, this was not the case as serum levels were the same or even higher in the non-responding FVB/N strain (0.6 μM) and in rabbits (8.4 μM), compared to the highly responding C57BL/6 mice (0.5 μM). In men, the miglustat level was >4 μM in serum and 8 μM in seminalplasma [13]

Our studies with the C57BL/6 × FVB/N interstrain hybrid mice provide an indication of the genetics of the sensitivity to miglustat. Whereas the majority of the fourth-generation hybrid mice responded to miglustat in a similar fashion as FVB/N mice, only very few displayed the C57BL/6 phenotype. About one-third of the interstrain hybrid mice simultaneouslyproduced spermatozoa with an acrosome and a falciform nuclear shape, as well as acrosome-deficient spermatozoa with grossly aberrant nuclei. Remarkably, these two types of spermatozoa were produced in various proportions that had not been seen in either of the parental strains. Clearly, the sensitivity of spermatogenesis for miglustat is not inherited in a Mendelian fashion. Rather, the reproductive impact of miglustat, expressed as the percentage of acrosome-less spermatozoa with a non-falciform nuclear shape, appears to be a quantitative trait. It is therefore likely that multiple genescontribute to the sensitivity of spermatogenesis to miglustat. Similar observations have been made in the study of the susceptibility to lunginjury by ozone. Mice of the A/J strain are susceptible in this respect, while C57BL/6 mice are resistant [23]. Recombinant inbred strains generated from these two strains displayed many intermediate levels of ozone susceptibility, in a continuous range, indicating a multigenic trait [24].

Competing interests

Authors' contributions

WB participated in the conception and design of the study, in the execution of experiments, analysis of experimental data, preparation of the data for publication, and in the writing of the manuscript. CMW participated in the execution of experiments, in the analysis of experimental data. MF, UV, EL and UG participated in the conception and design of the study and revised the manuscript critically. SB performed and analyzed western blotting experiments. FMP participated in the design of the study, and in writing of the manuscript. ACS participated in the conception and design of the study, in the execution of experiments, analysis of experimental data, preparation of the data for publication, and in the writing of the manuscript. All authorsread and approved the final manuscript.

The medical information provided on this website is of a general nature and can not substitute for the advice of a medical professional
(for example, a qualified doctor/physician)! Information from the internet could and should NOT be used to offer or render a medical opinion or otherwise
engage in the practice of medicine.